Method for the preparation of a viral vector by intermolecular homologous recombination

ABSTRACT

PCT No. PCT/FR95/01590 Sec. 371 Date Aug. 1, 1996 Sec. 102(e) Date Aug. 1, 1996 PCT Filed Dec. 1, 1995 PCT Pub. No. WO96/17070 PCT Pub. Date Jun. 6, 1996A recombinant adenoviral vector containing an exogenous DNA sequence insertion is prepared in a prokaryotic cell using homologous intermolecular recombination. The exogenous DNA sequence codes for a polypeptide of therapeutic interest for applications in gene therapy. The adenoviral vector may lack all or part of at least one region essential for replication.

The present invention relates to a method for preparing a viral vectorin vitro in a prokaryotic cell and to its application for producing aninfectious viral particle intended for therapeutic use, and especiallyfor use in gene therapy.

The possibility of treating human diseases by gene therapy has changedin a few years from the stage of theoretical considerations to that ofclinical applications. The first protocol applied to man was initiatedin the US in September 1990 on a patient who was geneticallyimmunodeficient as a result of a mutation affecting the gene coding foradenine deaminase (ADA). The relative success of this first experimentencouraged the development of new gene therapy protocols for variousgenetic or acquired diseases (infectious diseases, and viral diseases inparticular, such as AIDS, or cancers). The large majority of theprotocols described hitherto employ viral vectors to transfer thetherapeutic gene to the cells to be treated and to express it therein.

To date, retroviral vectors are among the ones most widely used onaccount of the simplicity of their genome. However, apart from theirrestricted capacity for cloning, they present two major drawbacks whichlimit their systematic use: on the one hand they chiefly infect dividingcells, and on the other hand, as a result of their integration at randomin the genome of the host cell, the risk of insertional mutagenesis isnot insignificant. For this reason, many scientific teams haveendeavored to develop other types of vector, among which thoseoriginating from adenoviruses, adeno-associated viruses (AAV),poxviruses and herpesviruses may be mentioned. Generally speaking, theirorganization and their infection cycle are amply described in theliterature available to a person skilled in the art.

In this connection, the use of adenoviral vectors has been seen to be apromising alternative. Adenoviruses have been demonstrated in manyanimal species, have a broad host range, have little pathogenicity anddo not present the drawbacks associated with retroviruses since theyreplicate in resting cells and are nonintegrative. As a guide, theirgenome consists of a linear, double-stranded DNA molecule ofapproximately 36 kb carrying more than about thirty genes, both earlygenes necessary for viral replication (E1 to E4) and late structuralgenes (L1 to L5) (see FIG. 1).

Generally speaking, the adenoviral vectors are obtained by deletion ofat least one portion of the viral genes (in particular of the E1 regionessential for viral replication), which are replaced by the therapeuticgenes. Consequently, they are propagated in a cell line, termedcomplementation line, which supplies in trans the deleted viralfunctions to generate a viral vector particle which is defective forreplication but capable of infecting a host cell. The line 293,established from human embryonic kidney cells, which complementsadenoviruses that are defective for the E1 function (Graham et al.,1977, J. Gen. Virol., 36, 59-72), is commonly used.

The techniques of preparation of adenoviral vectors are amply describedin the literature (see, in particular, Graham and Prevec, Methods inMolecular Biology, Vol. 7; Gene Transfer and Expression Protocols; Ed:E. J. Murray, 1991, The Human Press Inc., Clinton, N.J.). One of themethods most often employed consists in generating the recombinantadenoviral vector in complementation cells transfected with a bacterialplasmid carrying the gene of interest subcloned within its adenoviralinsertion region and the adenoviral genome. Generally, the latter iscleaved with a suitable restriction enzyme so as to reduce theinfectivity of the parent viral DNA and to increase the efficiency ofisolation of the recombinant adenoviruses. However, a substantialbackground of infectious viral particles of parenteral origin isnevertheless observed, which necessitates an especially arduous task ofanalysis of the plaques obtained (arduous from a time and coststandpoint, since each plaque has to be amplified and analyzedindividually). This is problematical when the parent virus displays aselective advantage over the recombinant adenovirus, for example whenthe latter replicates more slowly than the parent virus as a result ofthe insertion of a large-sized gene of interest (factor VIII,dystrophin), reducing proportionately the probability of obtaining it.

Ligation between two DNA fragments generated by the standard techniquesof molecular biology and carrying, respectively, the 5' and 3' portionsof the recombinant adenoviral vector may also be employed. Transfectionof the ligation mixture into the complementation cells makes it possiblein theory to encapsidate the genome of the recombinant adenovirus toform an infectious particle. This technology is of low efficiency andits application limited by the restricted number of suitable and uniquerestriction sites.

It has now been shown to be possible to generate a recombinantadenoviral vector in Escherichia coli (E. coli) by intermolecularhomologous recombination between a plasmid containing the genome of atype 5 adenovirus and an insert carrying an exogenous DNA sequencesurrounded by adenoviral sequences A and B (FIG. 2). This method leadsto the replacement in the adenoviral genome of the targeted regionlocated between A and B by the exogenous sequence. Transfection of therecombinant adenoviral vector thus generated into an appropriatecomplementation line gives rise to an infectious viral particle whichmay be used without a prior purification step to infect a host cell. Thebackground (contamination with parent viral particles) is reduced oreven abolished. In addition, it was found, surprisingly, that the use ofE. coli recBC sbcBC strains is especially advantageous for promotingintermolecular recombination between any two DNA fragments.

The method of the present invention is based on exploitation of theendogenous enzymatic activities of the prokaryotic cells involved inhomologous recombination. This intermolecular recombination techniquehad already been described for the cloning of small inserts intobacterial vectors (Bubeck et al., 1993, Nucleic Acids Research, 21,3601-3602) or for generating hybrid genes by intramolecularrecombination (Calogero et al., 1992, FEMS Microbiology Letters, 97,41-44; Caramori et al., 1991, Gene, 98, 37-44). However, thisrecombination technology had never been employed in prokaryotic cells togenerate infectious viral vectors (capable of being encapsidated intoinfectious viral particles).

The method of the invention has the advantage over the previoustechniques of being rapid and especially efficient, and of requiringonly a small number of manipulations in vitro. Furthermore, it may beemployed with DNA fragments generated by PCR (polymerase chainreaction), thereby avoiding the steps of subcloning of the exogenous DNAsequence into the insertion region of the viral genome. Lastly, itprovides an advantageous solution to the problem of background which isclearly identified in the literature. Consequently, it proves especiallyefficacious for vectors originating from large-sized viruses or virusesinto which the insertion of a large-sized exogenous DNA sequence isenvisaged.

Accordingly, the subject of the present invention is a method forpreparing, in a prokaryotic cell, a recombinant viral vector derivedfrom a parent virus into the genome of which an exogenous DNA sequenceis inserted, by intermolecular recombination between (i) a first DNAfragment comprising all or part of said genome of the parent virus and(ii) a second DNA fragment comprising said exogenous DNA sequencesurrounded by flanking sequences A and B which are homologous to (i).

For the purposes of the present invention, a recombinant viral vector isobtained from a parent virus modified so as to carry at a suitable siteof the viral genome and to express an exogenous DNA sequence. The parentviruses capable of being used in the context of the invention are, forexample, alphaviruses, retroviruses, poxviruses, and in particularvaccinia or canarypox virus, herpesviruses, adeno-associated viruses(AAV) and most especially adenoviruses.

In this connection, the choice of parent adenovirus is very wide. It canbe an adenovirus of human, canine, avian, bovine, murine, ovine, porcineor simian origin, or alternatively a hybrid adenovirus comprisingadenoviral genome fragments of different origins. Very specialpreference will be given to a serotype C adenovirus of human origin andpreferably a type 2 or 5 adenovirus, or alternatively to an adenovirusof animal origin of the CAV-1 or CAV-2 (canine), DAV (avian) oralternatively BAd (bovine) type. These viruses and their genome aredescribed in the literature (see, for example, Zakharchuk et al., 1993,Arch. Virol., 128, 171-176; Spibey and Cavanagh, 1989, J. Gen. Virol.,70, 165-172; Jouvenne et al., 1987, Gene, 60, 21-28; Mittal et al.,1995, J. Gen. Virol., 76, 93-102).

The objective of a method according to the invention is to prepare arecombinant viral vector for the transfer of an exogenous DNA sequenceto a host cell and its expression therein. "Exogenous DNA sequence" isunderstood to mean a nucleic acid which comprises coding sequences andregulatory sequences permitting the expression of said coding sequences,and in which the coding sequences are sequences which are not normallypresent in the genome of a parent virus employed in the presentinvention or, if they are present, are in a different genomic context.In the context of the invention, the exogenous DNA sequence may becomposed of one or more genes. The regulatory sequences may be of anyorigin.

Preferably, the exogenous DNA sequence can code for an antisense RNAand/or an mRNA which will then be translated into a protein of interest.It may be of the genomic type, of the complementary DNA (cDNA) type orof mixed type (minigene, in which at least one intron is deleted). Itmay code for a mature protein, a precursor of a mature protein, inparticular a precursor intended to be secreted and, as a result,comprising a signal peptide, a chimeric protein originating from thefusion of sequences of diverse origins or a mutant of a natural proteindisplaying improved or modified biological properties. Such a mutant maybe obtained by mutation, deletion, substitution and/or addition of oneor more nucleotide(s) of the gene coding for the natural protein.

In the context of the present invention, it can be advantageous to use:

the genes coding for cytokines or lymphokines, such as interferons α, βand γ, interleukins, and in particular IL-2, IL-6 or IL-10, tumornecrosis factors (TNF) and colony stimulating factors (CSF);

the genes coding for cell receptors, such as the receptors recognized bypathogenic organisms (viruses, bacteria or parasites), preferably by theHIV virus (human immunodeficiency virus), or ligands for cell receptors;

the genes coding for growth hormones (HGF);

the genes coding for coagulation factors, such as factor VIII and factorIX;

the gene coding for dystrophin or minidystrophin;

the gene coding for insulin;

the genes coding for polypeptides participating directly or indirectlyin cellular ion channels, such as the CFTR (cystic fibrosistransmembrane conductance regulator) protein;

the genes coding for antisense RNAs or proteins capable of inhibitingthe activity of a protein produced by a pathogenic gene present in thegenome of a pathogenic organism, or by a cellular gene whose expressionis deregulated, for example an oncogene;

the genes coding for a protease inhibitor such as α₁ -antitrypsin or aninhibitor of a viral protease;

the genes coding for variants of pathogenic proteins which have beenmutated in such a way as to impair their biological function, such as,for example, trans-dominant variants of the HIV virus TAT protein whichare capable of competing with the natural protein for binding to thetarget sequence, thereby preventing the replication of HIV;

the genes coding for antigenic epitopes so as to increase the immunityof the host cell;

the genes coding for polypeptides having anticancer properties, and inparticular tumor suppressors such as the p53 protein;

the genes coding for proteins of the major histocompatibility complexclasses I and II, as well as the genes coding for the proteins thatregulate the expression of these genes;

the genes coding for cellular enzymes or those produced by pathogenicorganisms; and

suicide genes. The HSV-1 TK gene may be mentioned more especially. Theviral TK enzyme displays a markedly greater affinity than the cellularTK enzyme for certain nucleoside analogs (such as acyclovir organciclovir). It converts them to monophosphated molecules, which arethemselves convertible by the cellular enzymes to nucleotide precursors,which are toxic. These nucleotide analogs can be incorporated in DNAmolecules in the process of synthesis, hence mainly in the DNA of cellsundergoing replication. This incorporation enables dividing cells suchas cancer cells to be destroyed specifically.

the genes coding for an antibody, an antibody fragment or animmunotoxin.

reporter genes such as the LacZ gene coding for β-galactosidase, or theluciferase gene.

This list is not limiting, and other genes may naturally be employed aswell.

Moreover, an exogenous DNA sequence employed in the present inventionmay comprise, in addition, a nontherapeutic gene, for example a genecoding for a selectable marker enabling the transfected host cells to beselected or identified. There may be mentioned the neo gene (coding forneomycin phosphotransferase) conferring resistance to the antibioticG418, the dhfr (dihydrofolate reductase) gene, the CAT (chloramphenicolacetyltransferase) gene, the pac (puromycin acetyltransferase) gene oralternatively the gpt (xanthine:guanine phosphoribosyltransferase) gene.

Naturally, a gene employed in the present invention may be placed underthe control of elements suitable for its expression in a host cell."Suitable elements" are understood to mean the set of elements necessaryfor its transcription into RNA (antisense RNA or mRNA) and for thetranslation of an mRNA into protein. Among elements necessary fortranscription, the promoter assumes special importance. The latter canbe a constitutive promoter or a regulable promoter, and it may beisolated from any gene of eukaryotic or even of viral origin.Alternatively, it can be the natural promoter of the gene in question.Generally speaking, a promoter employed in the present invention may bemodified so as to contain regulatory sequences. As examples oftissue-specific promoters, there may be mentioned those of theimmunoglobulin genes when it is sought to target the expression intolymphocytic host cells, and of the α₁ -antitrypsin gene for aliver-specific expression. The constitutive promoters of the HSV-1 TK(herpesvirus type 1 thymidine kinase) and of the murine or human PGK(phosphoglycerate kinase) gene, the SV40 (simian virus 40) earlypromoter, a retroviral LTR or alternatively the adenoviral MLP promoter,in particular of human adenovirus type 2, may also be mentioned.

The method according to the invention employs an intermolecularhomologous recombination mechanism. Generally speaking, it consists ofthe exchange of homologous sequences between two DNA fragments. Thesesequences may be identical or substantially homologous. In other words,the degree of homology of the sequences A and B with the correspondingportion of the first DNA fragment may be variable, but must besufficient to permit intermolecular recombination. For the purposes ofthe present invention, it is preferable for it to be greater than 70%,advantageously greater than 80%, preferably greater than 90%, and as anabsolute preference in the region of 100%. Furthermore, a short regionof homology may be sufficient (at least 10 consecutive nucleotides incommon between the sequences A and B and their homologous sequences inthe first DNA fragment). In the context of the present invention, thelength of the sequences A and B is preferably between 10 bp and 10 kb,advantageously 20 bp and 5 kb, preferably 30 bp and 2 kb, and as anabsolute preference 40 bp and 1 kb.

According to an advantageous embodiment, a method according to theinvention leads to the replacement of the genetic material localizedbetween the sequences of the first DNA fragment which are homologous tothe sequences A and B by the exogenous sequence. This intermolecularexchange enables a circular recombinant viral vector to be generated inthe form of a prokaryotic vector (plasmid, cosmid or phage) permittingits manipulation and/or its propagation in the prokaryotic cell. Anembodiment of the mechanism employed in the context of the presentinvention is illustrated in FIG. 2.

Although the insertion region of the exogenous sequence may be locatedin any position of the viral genome, it is preferable to avoid theregions acting in cis necessary for replication. These regions comprise,in particular, the 5' and 3' LTRs as well as the encapsidation signal inthe case of retroviruses, and the 5' and 3' ITRs and the encapsidationregion as regards adenoviruses. It would appear that the insertionregion may be directed into a variety of positions in accordance withthe chosen homologous sequences A and B.

As stated above, the first DNA fragment comprises all or part of thegenome of the parent virus employed in the context of the invention.This may be the genome of a wild-type virus or the genome of a virusderived therefrom, modified by deletion, addition and/or substitution ofone or more nucleotides. In particular, one or more genes may be whollyor partially deleted from the genome in question.

The first DNA fragment is preferably included in a conventional vector.The choice of the latter is very wide, but pBR322, p polyII oralternatively p polyIII*I (Lathe et al., 1987, Gene, 57, 193-201) may bementioned more especially. According to an advantageous embodiment ofthe method according to the invention, the first DNA fragment ispreferably linearized in the region in which the targeting of theinsertion is envisaged. In particular, it may be cleaved with one ormore restriction enzymes whose sites are localized between the sequencesof the first DNA fragment which are homologous to the sequences A and B,but also within these latter, although this embodiment is not preferred.The choice of restriction sites to be used according to the parent virusadopted is within the capacity of a person skilled in the art.Advantageously, it will be preferable to use sites which are notstrongly represented in the first DNA fragment, and especially uniquesites. Moreover, such sites may also be created by the standardtechniques of directed mutagenesis.

The second DNA fragment employed in the present invention carries, inparticular, the exogenous DNA sequence surrounded by the sequences A andB involved in the intermolecular recombination. It is preferable toemploy a linear DNA fragment. It may be generated by PCR, excised fromany vector or produced synthetically or by any conventional method inthe field of the art. As an example, a second DNA fragment employed inthe context of the present invention may be obtained by amplification ofthe exogenous DNA sequence from a vector of the prior art or from agenomic library or from an appropriate cDNA using suitable primersequipped at their 5' ends with the sequences A and B. In addition, asecond DNA fragment may also comprise plasmid sequences at its 5' and 3'ends, which may optionally be used by way of sequences A and B providedthey are homologous to the plasmid sequences contained in the first DNAfragment.

In accordance with the objectives pursued by the present invention, amethod according to the invention is carried out for the preparation ofa recombinant viral vector which is defective for replication. This termdenotes a vector incapable of completing an autonomous infectious cyclein a host cell. Generally, the genome of these defective vectors lacksone or more genes essential for replication, early genes and/or lategenes. They may be wholly or partially deleted or rendered nonfunctionalby mutation (addition, deletion and/or substitution of one or morenucleotides). In this connection, a method according to the inventioncan enable a recombinant adenoviral vector lacking all or part of theE1, E2, E3 and/or E4 regions to be prepared.

By way of illustration, the following embodiments may be mentioned. Whenthe intention is to prepare, in a prokaryotic cell, a recombinantadenoviral vector derived from a type 5 human adenovirus (Ad5) in whichthe insertion of an exogenous DNA sequence in place of the El region isenvisaged, it is possible to employ (i) a vector containing the Ad5genome cleaved with the enzyme ClaI (restriction site located atposition 918 of the Ad5 genome, as disclosed in the Genebank databankunder the reference M73260), and (ii) a DNA fragment comprising asequence A homologous to the adenoviral encapsidation region, theexogenous DNA sequence followed by a sequence B homologous to thesequence coding for the pIX protein. Moreover, the use of a vectorcarrying the adenoviral genome cleaved with the enzyme SpeI (position27082 of the Ad5 genome) and of a DNA fragment comprising a sequence Ahomologous to the 5' end of the E2 region, the exogenous DNA sequenceand a sequence B homologous to the 3' end of the E4 region will enable arecombinant adenoviral vector to be prepared in which the exogenous DNAsequence is inserted in place of the E3 region. Naturally, theseparticular embodiments are mentioned only as examples. Lastly, themethod according to the invention may be used to introduce deletions,mutations and/or substitutions into a particular region of a viralgenome.

The present invention proves especially advantageous when the intentionis to prepare a recombinant viral vector of at least 20 kb, and as anabsolute preference of at least 30 kb, and more especially in the caseof a vector having a genome whose length is from 80 to 120% of that ofthe genome of the corresponding wildtype virus, in particular from 90 to110% and preferably from 95 to 105%.

According to another embodiment, a method according to the invention mayalso be employed to insert at least two DNA fragments within the viralgenome, by intermolecular recombination between (i) a first DNA fragmentcomprising all or part of said genome of the parent virus, (ii) a secondDNA fragment comprising a first portion of said exogenous DNA sequenceequipped at its 5' end with said flanking sequence A and (iii) a thirdDNA fragment comprising a second portion of said exogenous DNA sequenceequipped at its 3' end with said flanking sequence B; said second andthird DNA fragments containing a homologous sequence overlapping attheir respective 3' and 5' ends. As a guide, these sequences which arehomologous between the second and third DNA fragments satisfy the samecriteria of homology and of length as the sequences A and B. Thisspecific embodiment is especially advantageous for the cloning oflarge-sized exogenous sequences.

A method according to the invention may be carried out in anyprokaryotic cell, and in particular in a bacterium derived from anEscherichia coli strain. However, it is most especially preferable toemploy a recBC sbcBC strain such as, for example, the strains CES200,CES201, W5449 and BJ5183 (Hanahan, 1983, J. Mol. Biol., 166, 557-580).

A procedure typical of a method according to the invention comprises thefollowing steps:

(a) a first DNA fragment and (ii) a second DNA fragment, as are definedabove, are cointroduced or introduced separately into a prokaryoticcell,

(b) the prokaryotic cell obtained in step (a) is cultured under suitableconditions to permit the generation of the recombinant viral vector byintermolecular recombination, and

(c) the recombinant viral vector is recovered.

In the context of a method according to the invention, the amounts offirst and second DNA fragments may vary. It is preferable to employ aconcentration of second fragment which is 10 times as large as that ofthe first fragment. The introduction of the DNA fragments into aprokaryotic cell and the recovery of the vector from these same cellsare carried out according to the general techniques of geneticengineering and of molecular cloning detailed in Maniatis et al. (1989,Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold SpringHarbor, N.Y.).

The present invention also relates to a method for preparing aninfectious viral particle containing a recombinant viral vector obtainedby carrying out a method according to the invention, according to which:

(a) said recombinant viral vector is introduced into a mammalian cell togenerate a transfected mammalian cell,

(b) said transfected mammalian cell is cultured under suitableconditions to permit the production of said viral particle, and

(c) said viral particle is recovered from the cell culture obtained instep (b).

The cells may be transfected according to the standard techniques wellknown to a person skilled in the art. The calcium phosphate technique,the DEAE-dextran technique, electroporation, methods based on osmoticshock, microinjection or methods based on the use of liposomes may bementioned in particular. According to a preferred embodiment, themammalian cell is advantageously a complementation cell, and inparticular the 293 cell in the context of a vector derived from anadenovirus. The viral particles may be recovered from the culturesupernatant, but also from the cells according to conventionalprotocols.

The present invention also covers the use of an infectious viralparticle or of a recombinant viral vector prepared according to a methodaccording to the invention, for the therapeutic or surgical treatment ofthe human body, and in particular by gene therapy. A method according tothe invention is intended more especially for the preventive or curativetreatment of diseases such as genetic diseases (hemophilia;thalassemias, emphysema, Gaucher's disease, cystic fibrosis, Duchenne'sor Becker's myopathy, etc.), cancers and viral diseases (AIDS, herpesinfections or infections caused by cytomegalovirus or bypapilloma-virus). For the purposes of the present invention, the vectorsand viral particles prepared by a method according to the invention maybe introduced either in vitro into a host cell removed from the patient,or directly in vivo into the body to be treated. Preferably, the hostcell is a human cell, and preferably a lung, fibroblast, muscle, liveror lymphocytic cell or a cell of the hematopoietic line.

The present invention also relates to a pharmaceutical compositioncomprising a therapeutically effective amount of an infectious viralparticle or of a recombinant viral vector prepared according to a methodaccording to the invention, in combination with a vehicle which isacceptable from a pharmaceutical standpoint. Such a pharmaceuticalcomposition may be prepared according to the techniques commonlyemployed and administered by any known administration route, for examplesystemically (in particular intravenously, intratracheally,intraperitoneally, intramuscularly, subcutaneously, intratumorally orintracranially) or by aerosolization or intrapulmonary instillation.

Lastly, the subject of the present invention is also the use of aninfectious viral particle or of a recombinant viral vector preparedaccording to a method according to the invention, for the expression ofa DNA sequence of interest in a cell system.

The present invention is described more completely by reference to thefigures which follow and by means of the examples which follow.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 is a diagrammatic representation of the human adenovirus type 5genome (shown in arbitrary units from 0 to 100), indicating the locationof the different genes.

FIG. 2 illustrates a method of intermolecular recombination between twoDNA fragments. The plasmid sequences are represented by a thin line, theviral sequences by a thick line and the exogenous sequence by a shadedbox.

FIG. 3 is a diagrammatic representation of the vector pTG3614,corresponding to p polyII into which is cloned the genome of arecombinant adenovirus modified in the E1 region by insertion of acassette for the expression of the human FIX gene.

FIG. 4 is a diagrammatic representation of the vector pTG4652,corresponding to p polyII into which is cloned the genome of arecombinant adenovirus modified in the E1, E3 and E4 regions byinsertion of a cassette for the expression of the human CF gene andpartial deletions.

EXAMPLES

The examples which follow illustrate just one embodiment of the presentinvention.

The constructions described below are carried out according to thegeneral techniques of genetic engineering and molecular biology detailedin Maniatis et al. (1989, supra). 5' protruding restriction sites may beconverted to blunt sites by treatment with the large Klenow fragment ofE. coli DNA polymerase, whereas 3' protruding sites are treated with T4polymerase. As regards the steps of PCR amplification, the protocol asdescribed in PCR Protocols--A guide to methods and applications (1990,ed. Innis, Gelfand, Sninsky and White, Academic Press Inc.) is applied.Cells are transfected by conventional methods and are cultured accordingto the supplier's recommendations. The fragments inserted into thedifferent constructions described below are indicated preciselyaccording to their position in the Ad5 genome as disclosed in theGenebank databank under the reference M73260.

EXAMPLE 1 Construction of a Recombinant Viral Vector Derived From a Tope5 Human Adenovirus in the Form of a Bacterial Plasmid

A. Cloning of the adenovirus 5 (Ad5) genome into p polyII

The left-hand end of the Ad5 genome (nucleotides 1 to 935) issynthesized by PCR using the oligonucleotide primers oTG6597 (SEQ IDNO: 1) and oTG6598 (SEQ ID NO: 2). The first primer hybridizes with thefirst 21 nucleotides of the 5' ITR, and possesses a PacI siteimmediately upstream of the viral sequences and an EcoRI site used forthe cloning. The second primer enables SalI and then BglII sites to beintroduced downstream of the viral sequences. The template utilized inthis reaction is Ad5 genomic DNA prepared according to conventionalmethods. The fragment thus amplified is digested with EcoRI and BglII[sic] and cloned into the plasmid p polyII (Lathe et al., 1987, supra)previously cleaved with these same two restriction enzymes, to givepTG1692.

The DNA fragment corresponding to the right-hand end of the Ad5 genome(nucleotides 35103 to 35935) is prepared according to the sameprinciple, using the oligonucleotide pair oTG6599 (SEQ ID NO: 3) andoTG6597 (SEQ ID NO: 1). pTG1693 is constructed by inserting theamplified fragment, digested with EcoRI and BglII, into the same sitesof p polyII. A BglII-BamHI fragment carrying the amplified sequences isexcised from pTG1693 so as to introduce it into the BglII site ofpTG1692 downstream of the adenoviral 5' sequences. pTG3601 therebyobtained is linearized between the left-hand and right-hand ends of theAd5 genome by digestion with restriction enzymes BglII or SalI. The endsthus generated are blunted by the action of the Klenow fragment of E.coli DNA polymerase I. BJ5183 bacteria (Hanahan, 1983 supra), madecompetent by calcium chloride treatment, are cotransformed with theabove preparation and the Ad5 genomic DNA. The colonies obtained areanalyzed by restriction mapping. One clone is selected, designatedpTG3602, generated by intermolecular homologous recombination, in whichthe adenoviral sequences (nucleotides 936 to 35102) have been insertedbetween the two fragments produced by PCR, so as to generate a plasmidvector comprising the complete Ad5 genome-pTG3602 is produced in C600bacteria (Huynh et al., 1985 DNA cloning, Volume 1, ed. Glover, IRLPress Limited: Oxford, England pp. 56110).

B. Evaluation of the infectious power of pTG3602

The viral genome is liberated by the action of the restriction enzymePacI. 293 cells cultured in a monolayer are transfected with the productof this digestion by the calcium phosphate precipitation technique. Itis also possible to use other sensitive cells, for example A549 cells(ATCC CCL185). The cells are grown under agar for 9 to 14 days, duringwhich period the appearance of lytic plaques on the cell lawn testifiesto the production of infectious viral particles, and hence to thecapacity of the Ad5 genome originating from pTG3602 to replicate.

C. Construction of a defective recombinant adenoviral vector in whichthe exogenous gene replaces the E1 early region.

A plasmid is used in which the LacZ reporter gene coding forβ-galactosidase is placed in a viral context; for example a plasmidderived from pMLP-Adk7 (Stratford-Perricaudet and Perricaudet, 1991,Human Gene Transfer, 219, 51-61) comprising the Ad5 5' sequences(nucleotides 1 to 458), the Ad2 MLP promoter, the LacZ gene, the SV40virus polyadenylation signal and the Ad5 sequences lying betweennucleotides 3329 and 6241.

The LacZ gene expression cassette surrounded by adenoviral sequences isexcised by the action of the restriction enzymes BsrGI (position 192 onthe Ad5 genome) and PstI (position 3788) and then purified. pTG3602 islinearized at the ClaI site (position 918) and then treated with Klenow.The two fragments obtained are cotransformed into competent BJ5183bacteria. Recombination at the homologous adenoviral sequences bringsabout the replacement of the Ad5 E1 region (nucleotides 459 to 3328) bythe expression cassette for the reporter gene in the plasmid pTG3603.

Its infectious power is verified by transfection of a lawn of 293 cellstransfected with pTG3603 previously digested with PacI. The lyticplaques formed are then picked out and the viral particles resuspendedand used to infect a monolayer of 293 cells. The blue coloration of thecells after addition of X-Gal testifies to the expression of the LacZgene.

D. Construction of a recombinant adenoviral vector in which theexogenous gene replaces the E3 early region.

A cassette for the expression of the gene coding for the gp19 protein ofthe Ad5 E3 region (nucleotides 28731 to 29217) is assembled in thebacteriophage M13mp18 (Gibco BRL) by cloning two PCR fragments, onecorresponding to the RSV (Rous sarcoma virus) 3' LTR (oligonucleotideprimers oTG5892-SEQ ID NO: 4 and 5893-SEQ ID NO:5) and the other to thesequence coding for gp19 (oTG5455-SEQ ID NO:6 and 5456-SEQ ID NO:7). Thevector M13TG1683 is obtained, from which the expression cassette isexcised by an XbaI digestion. After treatment with Klenow, it isintroduced into the BsmI site (blunted by the action of phage T4 DNApolymerase) of pTG8519. The latter is derived from the plasmid puc19(Gibco BRL), into which the adenoviral sequences lying between the SpeIsite and the right-hand end of the genome (nucleotides 27082 to 35935)but lacking the majority of the E3 region (nucleotides 27871 to 30758)have been inserted. pTG1695 is obtained, the ScaI-SpeI fragment ofwhich, carrying the plasmid sequences, is replaced by a purifiedequivalent fragment of pTG1659. The latter corresponds to puc19 [sic]comprising the Ad5 sequences extending from nucleotides 21562 to 35826.pTG1697 thereby obtained possesses adenoviral sequences which extendfrom the BamHI (position 21562) site to the 3' ITR (position 35935), inwhich sequences the E3 region is replaced by a gp19 expression cassetteunder the control of the RSV constitutive promoter. the DraI fragment(position 22444 and 35142 on the Ad5 genome) is purified, andcointroduced into competent BJ-5183 bacteria with pTG3602 linearizedwith SpeI and treated with Klenow. The recombinants carrying a plasmidgenerated by recombination are screened for the presence of the RSVpromoter. pTG3605, a plasmid vector carrying the Ad-gp19+genome, is thusdemonstrated.

The infectious power viral genome excised from plasmid pTG3605 is testedaccording to the protocol already described above. The production of afunctional gp19 protein is monitored by co-immunoprecipitation of theantigens of the major histocompatibility complex class I and of theprotein (Burgert and Kvist, 1985, Cell, 41, 987-997).

E. Construction of a defective recombinant adenoviral vector in whichboth early regions E1 and E3 are replaced by exogenous genes BJ-5183cells are cotransformed with the DraI fragment isolated from pTG1697mentioned above, and the SpeI fragment (Klenow) isolated from pTG3603.The resulting plasmid is tested under the same conditions as above.

F. Construction of a recombinant adenoviral vector expressing the FIXgene

The cDNA coding for human FIX was cloned in the form of a BamHI fragmentinto the plasmid pTG381. In the latter, the 5' non-coding end of the FIXgene was modified so as to comply with Kozak's rules. Plasmid pTG381 isdescribed in Patent Publication FR 2,600,334. The FIX gene is reclonedinto the BamHI site of pTG6512 to give pTG4375. pTG6512 is derived fromthe vector pTG6511 after deletion of the Ad2 major late promoter (MLP)and replacement by a polylinker. As a guide, the construction of pTG6511is described in detail in Example 1 of International Application WO94/28152. The murine PGK promoter is isolated by conventional methodsfrom mouse genomic DNA according to Adra et al. (1987, Gene 60, 65-74),or by means of appropriate primers created from the data of the sequencedisclosed in the Genebank databank under the reference X15339. PstIrestriction sites are included at the 5' ends of the primers in order tofacilitate the subsequent cloning steps. The fragment carrying the PGKpromoter is treated with T4 polymerase and then introduced upstream ofthe human FIX gene into the HpaI site of pTG4375 from which a so-called"replacement" fragment, containing the FIX cassette surrounded by E1adenoviral sequences (185 bp at the 5' end and 2045 bp at the 3' end),is excised by MscI digestion.

The latter fragment is cotransformed into BJ5183 bacteria in thepresence of the vector pTG3602 digested with ClaI (position 918). Thetransformants are selected on ampicillin and analyzed by restrictionmapping. The clone designated pTG3614, corresponding to an adenoviralvector containing the FIX therapeutic gene in place of the E1 region(FIG. 3), is selected. A viral stock is constituted in a conventionalmanner, by transfection of the adenoviral genome liberated by PacIdigestion into the 293 line. After an amplification step, the cells arelysed and the AdTG3614 viral particles purified by centrifugation oncesium chloride.

The capacity to transfer and express the FIX gene is evaluated in amouse animal model. For this purpose, 1.5×10⁹ pfu are injected into thecaudal vein of 5- to 6-week-old female C57 Black 6 mice. Blood samplesare drawn regularly, on which the amount of human FIX is assayed byELISA (Diagnostica Stago Kit, Asnieres, France; AsserachiromR IX: Ag00564). FIX is detected in the serum of mice for several weeks with amaximum at 5 days.

G. Construction of an adenoviral vector modified in the E2 region

During the normal viral cycle, the expression of the genes of the E2region is induced by the early products of E1 and E4, and a copiousproduction of the proteins encoded by E2, and especially of the DBPprotein (for DNA binding protein), is observed. In point of fact, thishigh expression can be problematical in vivo (induction of inflammatoryresponses or interference with the expression of the transgene). Forthis reason, adenoviral vectors defective for the E2A function wereconstructed, either by introduction of a temperature-sensitive mutationinto E2A (position 22795; C to T resulting in a change from Pro to Ser)or by a partial deletion of the DBP gene.

The vector pTG9551 carrying the temperature-sensitive mutation isgenerated by homologous recombination in BJ cells between pTG3601digested with BglII and the purified genomic DNA of the Ad5Hts 125 virus(Ensinger and Ginsberg, 1972, J. Virol. 10, 328-339). The genome thusreconstituted is liberated by PacI digestion and transfected into 293cells, which are incubated at 32° C. for the production of the viralparticles (the non-permissive temperature is 39° C.).

The defective E2A character may also be generated by partial deletion ofthe coding regions of the DBP gene, avoiding the regions covering otherreading frames. The construction is carried out by homologousrecombination between pTG3601 BglII and the viral DNA prepared from theH5d1802 virus (Rice and Klessig, 1985, J. Virol. 56, 767-778). Theviruses may be produced in a suitable complementation linetranscomplementing the DBP function, for example line 293 transfectedwith a vector permitting the constitutive or regulated expression of theDBP gene.

H. Construction of an adenoviral vector modified in the E4 region

In a first stage, a so-called "replacement" vector is constructed,comprising a cassette for the expression of the gene coding for the CFTRprotein placed within the E1 adenoviral region. This vector, designatedpTG8585, contains the following elements, all described in theliterature:

the Ad5 5' ITR (positions 1 to 458),

the Ad2 MLP promoter,

human CFTR cDNA,

the polyadenylation signal of the rabbit β-globin gene,

the RSV virus (Rous sarcoma virus) 3' LTR,

the adenoviral sequences coding for the gp19 protein (positions 28731 to29217 of Add5), and

the 3' portion of the E1B region (position 3329 to 6241).

In parallel, the adenoviral sequences covering the E3 and E4 regions aresubcloned into the vector p polyII and deleted by the standardtechniques of molecular biology. The E3 sequences (positions 28592 to30470) are removed by XbaI digestion and the E4 sequences (positions32994 to 34998) are removed. The adenoviral genome thus modified isreconstituted by homologous recombination between a replacement fragmentcarrying these modifications, isolated from the above vector, andpTG3603 digested with SpeI. pTG8595 is obtained, carrying a recombinantadenoviral genome (LacZ gene in place of the E1 region) which isdefective for the E3 and E4 functions.

The cassettes for the expression of the CFTR and gp19 genes, describedabove, are introduced into pTG8595 as a replacement for the LacZ gene,by cotransformation of BJ5183 cells with the PacI-BstEII fragmentisolated from pTG8585 and the vector pTG8595 linearized with ClaI.pTG4652 (FIG. 4) is generated, which may be propagated in a cell linecomplementing the defective E1 and E4 functions as are described inInternational Application WO94/28152.

The viral particles are amplified, the cells are lysed and the totalcell extracts are analyzed for the expression of the CFTR gene byWestern blotting (8% SDSPAGE gel). The filters are incubated in thepresence of a 1/5000 dilution of the monoclonal antibody MAB 1104directed against a synthetic peptide corresponding to amino acids 722 to734 of the human CFTR protein. The detection is carried out by the ECL(enhanced chemiluminescence; Amersham kit) method. A band of strongintensity and rather diffuse, corresponding to a high molecular weightproduct comigrating with a CFTR control, is observed in the extracts ofcells infected with AdTG4652. This band is not present in the extractsoriginating from the parent virus pTG8595 which does not contain theCFTR expression cassette.

EXAMPLE 2 Construction of a Recombinant Viral Vector Derived From CanineAdenovirus 2 (CAV2) in the Form of a Bacterial Plasmid

A. Cloning of the CAV2 genome into p polyII

The CAV2 genome is cloned into p polyII SfiI-NotI 14 (Lathe et al.,1987, supra) and modified to generate recombinant vectors using theappropriate sites, according to the same methods as are used in the caseof the manipulation of Ad5. The left-hand end (nucleotides 1 to 810according to the numbering of the Genebank sequence D04368) andright-hand end (nucleotides˜27600 to the end) are synthesized by PCRbetween the oligois nucleotide primer pairs oTG6974 (SEQ ID NO: 8) andoTG6975 (SEQ ID NO: 9) and oTG6976 (SEQ ID NO: 10) and oTG6974,respectively. They are assembled via the PstI site introduced during thePCR following the viral sequences in the case of the left-hand end, andmapped at approximately 1200 bp from the end of the genome in the caseof the right-hand end. Competent BJ5183 bacteria are cotransformed withthe plasmid thereby obtained, linearized with PstI, and CAV2 DNA.Introduction of the missing viral sequences (nucleotides 811 to 27600)is carried out by homologous recombination. The CAV2 genome carried bythe resulting plasmid is excised via the NotI sites introduced duringthe PCR, and then transfected into a monolayer of MDCK cells (ATCCCCL34). The following steps are described in Example 1.

B. Construction of a recombinant adenoviral vector of canine origin

CAV2 virus (strain Toronto A 26/61; ATCC VR-800) genomic DNA is preparedby the standard technique (amplification on a dog kidney line MDCK GHK,etc., lysis of the cells, purification of the viruses by centrifugationon cesium chloride, treatment with proteinase k and lastlyphenol/chloroform extraction). The CAV2 genome, which is 31 kbp inlength, is introduced into a plasmid vector by homologous recombination.For this purpose, the left-hand and right-hand ends of the CAV2 genomeare isolated by PCR and enzymatic digestion, incorporating a NotI siteimmediately beyond the 5' and 3' ITRs. The vector pTG4457 is obtained byintroducing into p polyII the 5' portion of the viral genome as far asthe BstBI site (position 870) followed by the 3' portion starting fromthe SalI site (position 27800). The complete genome may be reconstitutedby cotransformation between the genomic DNA (100 to 500 ng) and pTG4457digested with BstBI (10 to 100 ng). The viral genome may be excised fromthe above vector pTG5406 by NotI digestion. It is verified that the DNAis infectious by transfection of 1 to 5 μg into dog MDCK or GHK cells.The production of plaques is observed.

The recombinant vector is obtained in the following manner:

The left-hand end of CAV2 is subcloned and modified so as to delete theE1A coding sequences in their entirety and the E1B coding sequencespartially. This is performed by NarI digestion, followed byreintroduction of a fragment amplified by PCR covering the encapsidationregion, the E1A promoter region and the transcription startsite. Theprimers are designed so as to integrate a unique XbaI restriction siteas well. pTG5407 is obtained, into which the CAT gene or LacZ gene(pTG5408) is introduced at the XbaI site. The latter plasmid is digestedwith XhoI, and the 3' portion of the viral genome is inserted in theform of a SalI fragment (position 27800 to the end of the 3' ITR). Thevector pTG5409 thereby obtained is linearized with SalI andcotransformed into E. coli BJ5183 with the CAV-2 genome digested withSwaI. An adenoviral vector of canine origin carrying the CAT reportergene in place of the El region is obtained.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 10                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 38                                                              <212> TYPE: DNA                                                               <213> ORGANISM: human adenovirus                                               - - <400> SEQUENCE: 1                                                         - - gccgaattct taattaacat catcaataat atacctta      - #                      - #     38                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 35                                                              <212> TYPE: DNA                                                               <213> ORGANISM: human adenovirus                                               - - <400> SEQUENCE: 2                                                         - - gacagatctg tcgacgtggc aggtaagatc gatca       - #                  -     #       35                                                                      - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 35                                                              <212> TYPE: DNA                                                               <213> ORGANISM: human adenovirus                                               - - <400> SEQUENCE: 3                                                         - - aggagatctg tcgactctca aacatgtctg cgggt       - #                  -     #       35                                                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 47                                                              <212> TYPE: DNA                                                               <213> ORGANISM: rous sarcoma virus                                             - - <400> SEQUENCE: 4                                                         - - gtcgtaggat ccagctgctc cctgcttgtg tgttggaggt cgctgag   - #                    47                                                                         - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 47                                                              <212> TYPE: DNA                                                               <213> ORGANISM: rous sarcoma virus                                             - - <400> SEQUENCE: 5                                                         - - gtagctgacg tcccaggtgc acaccaatgt ggtgaatggt caaatgg   - #                    47                                                                         - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: human adenovirus                                               - - <400> SEQUENCE: 6                                                         - - atcggaattc aagatgatta ggtac          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: human adenovirus                                               - - <400> SEQUENCE: 7                                                         - - atcgtctaga ttaaggcatt ttcttttc         - #                  - #                 28                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: canine adenovirus                                              - - <400> SEQUENCE: 8                                                         - - cagggatccg cggccgcatc atcaataata tacagg      - #                  -     #       36                                                                      - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: canine adenovirus                                              - - <400> SEQUENCE: 9                                                         - - ctgctgcagt cagaaatgct agcaggaga         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: canine adenovirus                                              - - <400> SEQUENCE: 10                                                        - - tgcggatcca cagactaagc ggaggta          - #                  - #                 27                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for preparing, in a prokaryotic cell, arecombinant adenoviral vector into the genome of which an exogenous DNAsequence is inserted, by intermolecular homologous recombinationcomprising the steps of(a) introducing into said prokaryotic cell:(i) afirst DNA fragment comprising all of said genome of the adenovirus orsaid adenoviral genome lacking all or part of the E1 region, E2 region,E3 region, E4 region or a combination thereof, and (ii) a second DNAfragment comprising said exogenous DNA sequence surrounded by flankingsequences A and B which are homologous to sequences of (i) not includingsequences of the 5'ITR, 3'ITR and encapsidation regions of saidadenoviral genome, such as to allow intermolecular homologousrecombination; wherein the first and second DNA fragments are notcarried by a single vector, and (b) culturing the prokaryotic cellobtained in (a) under suitable culture conditions to allow homologousrecombination and insertion of the exogenous DNA into the first DNAfragment to occur, and recovering the resulting recombinant adenoviralvector.
 2. The method according to claim 1, wherein the adenovirus is ofhuman, canine, avian, bovine, murine, ovine, porcine, or simian origin,or said adenovirus is a hybrid adenovirus.
 3. The method according toclaim 2, wherein the adenovirus is a type CAV-2 adenovirus of canineorigin.
 4. The method according to claim 2, wherein the adenovirus is aserotype C adenovirus of human origin.
 5. The method according to claim4, wherein the adenovirus is a type 5 adenovirus of human origin.
 6. Themethod according to claim 1, wherein said exogenous DNA sequence codesfor a polypeptide of therapeutic interest selected from the groupconsisting of coagulation factors, growth hormones, cytokines,lymphokines, tumor-suppressing polypeptides, cell receptors, ligands forcell receptors, protease inhibitors, antibodies, toxins, immunotoxins,dystrophin and polypeptides participating in cellular ion channels. 7.The method according to claim 1, wherein the homologous flankingsequences A and B are from 10 consecutive bp to 10 consecutive kb inlength.
 8. The method according to claims 1, wherein the first DNAfragment is linearized in the region where said exogenous sequence isinserted.
 9. The method according to claim 1, wherein said first DNAfragment lacks all or part of at least one region essential forreplication, selected from the group consisting of the E1, E2, E3 and E4regions and wherein the recombinant adenoviral vector is defective forreplication.
 10. The method according to claim 9, wherein therecombinant adenoviral vector lacks all or part of the E3 region. 11.The method according to claim 1, wherein the recombinant adenoviralvector is of at least 20 kb.
 12. The method according to claim 11,wherein the recombinant adenoviral vector is of at least 30 kb.
 13. Amethod for preparing, in a prokaryotic cell, a recombinant adenoviralvector into the genome of which an exogenous DNA sequence is inserted byintermolecular homologous recombination comprising the steps of(a)introducing into said prokaryotic cell:(i) a first DNA fragmentcomprising all of said genome of the adenovirus or said adenoviralgenome lacking all or part of the E1 region, E2 region, E3 region, E4region or a combination thereof, (ii) a second DNA fragment comprising afirst portion of said exogenous DNA sequence whose 5' end is equippedwith a flanking sequence A, and (iii) a third DNA fragment comprising asecond portion of said exogenous DNA sequence whose 3' end is equippedwith a flanking sequence B; said second and third DNA fragments furthercontaining an overlapping homologous sequence at their respective 3' and5' ends, and said flanking sequences A and B being homologous tosequences of (i) not including sequences of the 5'ITR, 3'ITR, andencapsidation regions of said adenoviral genome, such as to allow forintermolecular homologous recombination, and (b) culturing theprokaryotic cell obtained in (a) under suitable culture conditions toallow homologous recombination to occur, and recovering the resultingrecombinant adenoviral vector.
 14. The method according to claim 1,wherein said prokaryotic cell is a recBC sbcBC strain of Escherichiacoli.
 15. A method for preparing an adenoviral particle containing arecombinant adenoviral vector, according to which:(a) a recombinantadenoviral vector obtained according to method of claim 1 is introducedinto a mammalian cell to generate a transfected mammalian cell, (b) saidtransfected mammalian cell is cultured under suitable conditions topermit the production of said adenoviral particle, and (c) saidadenoviral particle is recovered from the cell culture obtained in step(b).
 16. A method for preparing in a prokaryotic cell, a recombinantadenoviral vector into the genome of which an exogenous DNA sequence isinserted by intermolecular homologous recombination comprising the stepsof:(i) preparing a first DNA fragment comprising all of said adenoviralgenome or said adenoviral genome lacking all or part of the E1 region,E2 region, E3 region, E4 region or any combination thereof, said firstDNA fragment being linearized in a region where insertion of saidexogenous DNA sequence is targeted,(ii) preparing a linear second DNAfragment comprising said exogenous DNA sequence and flanking sequences Aand B located at both sides of said linear second DNA fragment, and saidflanking sequences A and B being homologous to sequences of (i) notincluding sequences of the 5'ITR, 3'ITR and encapsidation regions ofsaid adenoviral genome, such as to allow for intermolecular homologousrecombination, (iii) introducing said first DNA fragment and second DNAfragment into said prokaryotic cell, (iv) culturing the prokaryotic cellobtained in step (iii) under suitable culture conditions, and (v)recovering the recombinant adenoviral vector.
 17. A method for modifyingin a prokaryotic cell, a particular region of an adenoviral vectorgenome by intermolecular homologous recombination comprising the stepsof(a) introducing into said prokaryotic cell:(i) a first DNA fragmentcomprising all of said adenoviral vector genome or said adenoviralvector genome lacking all or part of the E1 region, E2 region, E3region, E4 region or a combination thereof and (ii) a second DNAfragment comprising said particular adenoviral region of which one ormore nucleotides is modified by introduction of a deletion, addition,substitution, or any combination thereof, surrounded by flankingsequences A and B which are homologous to sequences of (i) not includingsequences of the 5'ITR, 3'ITR and encapsidation regions of saidadenoviral genome, such as to allow for intermolecular homologousrecombination,wherein the first and second DNA fragments are not carriedby a single vector, and (b) culturing the prokaryotic cell obtained in(a) under suitable culture conditions to allow homologous recombinationto occur, and recovering the resulting recombinant adenoviral vector.18. A method for modifying in a prokaryotic cell, a particular region ofan adenoviral vector genome by intermolecular homologous recombinationcomprising the steps of:(i) preparing a first DNA fragment comprisingall of said adenoviral vector genome or said adenoviral vector genomelacking all or part of the E1 region, E2 region, E3 region, E4 region ora combination thereof, said first DNA fragment being linearized in saidparticular region,(ii) preparing a linear second DNA fragment comprisingsaid particular region flanked by sequences A and B located at bothsides of said linear second DNA fragment, wherein said particular regionis modified by introduction of a deletion, addition, substitution, orany combination thereof, of one or more nucleotides and said flankingsequences A and B are homologous to sequences of (i) not includingsequences of the 5'ITR, 3'ITR and encapsidation regions of saidadenoviral genome, such as to allow for intermolecular homologousrecombination, (iii) introducing said first DNA fragment and second DNAfragment into said prokaryotic cell, (iv) culturing the prokaryotic cellobtained in step (iii) under suitable culture conditions, and (v)recovering the modified adenoviral vector.